Towards detection of structurally-diverse glycated epitopes in native proteins: Single-chain antibody directed to non-A1c epitope in human haemoglobin

Article

作者: Gaetani, Miss Lucia ; Rumary, Katherine L ; Tyreman, Matthew ; Davis, Paul ; Giorgi-Coll, Susan ; Pinto, Isabel Campos ; Li, Meng ; Schouten, James A ; Dixon, Ann M ; O'Connor, Peter

Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the β-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from β-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the β-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay.

2022-11-28·Journal of Chemical Information and Modeling

Do Go Chasing Waterfalls: Enoyl Reductase (FabI) in Complex with Inhibitors Stabilizes the Tetrameric Structure and Opens Water Channels

Article

作者: Maltarollo, Vinicius Gonçalves ; Shevchenko, Ekaterina ; Kronenberger, Thales ; Ferreira, Glaucio Monteiro ; Cino, Elio A. ; Lima, Igor Daniel de Miranda ; Poso, Antti

The enzyme enoyl-ACP reductase (FabI) is the limiting step of the membrane's fatty acid biosynthesis in bacteria and a druggable target for novel antibacterial agents. The FabI active form is a homotetramer, which displays the highest affinity to inhibitors. Herein, molecular dynamics studies were carried out using the structure of FabI in complex with known inhibitors to investigate their effects on tetramerization. Our results suggest that multimerization is essential for the integrity of the catalytic site and that inhibitor binding enables the multimerization by stabilizing the substrate binding loop (SBL, L:195-200) coupled with changes in the H4/5 (QR interface). We also observed that AFN-1252 (naphtpyridinone derivative) promotes unique conformational changes affecting monomer-monomer interfaces. These changes are induced by AFN-1252 interaction with key residues in the binding sites (Ala95, Tyr146, and Tyr156). In addition, the analysis of water trajectories indicated that AFN-1252 complexes allow more water molecules to enter the binding site than triclosan and MUT056399 complexes. FabI-AFN-1252 simulations show accumulation of water molecules near the Tyr146/147 pocket, which can become a hotspot to the design of novel FabI inhibitors.

2021-05-07·Science1区 · 综合性期刊

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

1区 · 综合性期刊

Article

作者: Mashhour, Aida Rahmani ; Rogers, Cromarte ; Wolf, Markus ; Fernández-García, Yaiza ; Günther, Christian ; Gribbon, Philip ; Uetrecht, Charlotte ; Boger, Juliane ; Dunkel, Ilona ; Hänle, Anna ; Usenik, Aleksandra ; Bento, Isabel ; Gieseler, Henry ; Meents, Alke ; Tolstikova, Alexandra ; Günther, Sebastian ; Bourenkov, Gleb ; Meyer, Jan ; Falke, Sven ; Lourdu Xavier, P. ; Schulz, Eike-Christian ; Andaleeb, Hina ; Reinke, Patrick Y. A. ; Brehm, Wolfgang ; von Stetten, David ; Saouane, Sofiane ; Schwinzer, Martin ; Knoska, Juraj ; Wollenhaupt, Jan ; Günther, Stephan ; Yefanov, Oleksandr ; Melo, Diogo ; Peña-Murillo, Gisel E. ; Trost, Fabian ; Krichel, Boris ; Ullah, Najeeb ; Gelisio, Luca ; Kuzikov, Maria ; Karničar, Katarina ; Lieske, Julia ; Niebling, Stephan ; Ewert, Wiebke ; Ginn, Helen M. ; Domaracky, Martin ; Cox, Russell ; França, Bruno Alves ; Werner, Nadine ; Groessler, Michael ; Ellinger, Bernhard ; Oberthuer, Dominik ; Garcia-Alai, Maria Marta ; Srinivasan, Vasundara ; Zaliani, Andrea ; Rarey, Matthias ; Schlünzen, Frank ; Han, Huijong ; Hinrichs, Winfried ; Beck, Tobias ; Pearson, Arwen R. ; Monteiro, Diana C. F. ; Pompidor, Guillaume ; Meier, Susanne ; Hilgenfeld, Rolf ; Lorenzen, Kristina ; Zhang, Linlin ; Sun, Xinyuanyuan ; Hennicke, Vincent ; Lane, Thomas J. ; Feiler, Christian G. ; Beccari, Andrea R. ; Seychell, Brandon ; Li, Chufeng ; Mehrabi, Pedram ; Fleckenstein, Holger ; Schubert, Robin ; Chapman, Henry N. ; Pletzer-Zelgert, Jonathan ; Tidow, Henning ; Koua, Faisal H. M. ; Fischer, Pontus ; Zaitseva-Doyle, Joanna J. ; Chari, Ashwin ; White, Thomas A. ; Schneider, Thomas R. ; Panneerselvam, Saravanan ; Peck, Ariana ; Ehrt, Christiane ; Kopicki, Janine-Denise ; Awel, Salah ; Betzel, Christian ; Hakanpää, Johanna ; Galchenkova, Marina ; Barthelmess, Miriam ; Norton-Baker, Brenna ; Escudero-Pérez, Beatriz ; Gevorkov, Yaroslav ; Loboda, Jure ; Schmidt, Christina ; Weiss, Manfred S. ; Karpics, Ivars ; Turk, Dusan ; Brognaro, Hévila

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^pro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^pro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

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